In recent years, opportunistic infectious diseases caused by viruses, such as cytomegalovirus (hereinafter abbreviated as CMV), in patients maintained on immunosuppressants, such as recipients of organ transplantation, have given rise to a problem. For instance, such infectious diseases have arisen the problem in the first living liver transplantation conducted in Shimane Medical Collage, Japan.
It has been clinically observed that the incidence of CMV infectious diseases varies depending on the combination of immunosuppressants administered to post-transplantation patients. Based on this observation, the present inventors studied the influences of individual immunosuppressants and combinations thereof on proliferation of CMV through in vitro testing systems. As a result, of various known immunosuppressants, cyclosporine and predonine accelerated CMV proliferation, while mizoribine and azathioprine inhibited CMV proliferation. These results are in good agreement with case reports as described below. Therefore, it is believed that substances exhibiting antiviral activity in vitro also possess antiviral activity in vivo.
More specifically, the cases have been reported in which CMV infection developed in 100% of patients on cyclosporine+predonine therapy whereas the incidence off CMV infection was as low as 51.5% or 63.6% in patients on the therapy with cyclosporine+predonine in combination with mizoribine or azathioprine, respectively, suggesting the contribution of mizoribine or azathioprine to the anti-CMV effect (see Shiraki K., et al., rinsho to virus, Vol. 18, pp. 25-29, "men-ekifuzenjotai ni okeru virus no saikasseika (Reactivation of virus in immunodeficiency)" (1990); Shiraki K., et al., Biomedica., vol. 5, pp. 65-69, "men-eki yokuseizai to CMV (Immunosuppressants and CMV)" (1990); Shiraki K., et al., Transplant. Proc., Vol. 22, pp. 1682-1685, "Effect of cyclosporine, azathioprine, mizoribine and predonine on replication of human cytomegalovirus" (1990); and Shiraki K., et al., Arch. Virol., Vol. 117, pp. 165-171, "Immunosuppressive dose of azathioprine inhibits replication of human cytomegalovirus" (1991).
The like observation applies to FK 506 and cyclosporine as immunosuppressants. It has been reported that FK 506 has no or slight inhibitory influence on CMV growth (see Shiraki K., et al., J. Antibiotics, Vol. 44, pp. 909-911, "Effect of FK 506 replication of human cytomegalovirus in vitro" (1991)), while cyclosporine reveals the above-mentioned results.
According to the clinical report of Alessiani, et al., no difference was recognized in the incidence of bacterial and fungal infectious diseases between liver transplantation recipients on FK 506+predonine therapy and those on cyclosporine+predonine therapy, while symptomatic CMV infectious disease developed in 0 out of 20 post-transplantation patients on the former therapy and 5 out of 20 post-transplantation patients on the latter therapy (see Alessiani M., et al., Transplant. Proc., Vol. 22, pp. 44-46, "Infection with FK 506 Immunosuppression; Preliminary results with primary therapy" (1990)). These clinical reports back up the CMV proliferation accelerating effect of cyclosporine observed in vitro.
Further, Bia, et al. made a case report that the incidence of CMV infection in post-transplantation patients on azathioprine+steroid therapy was about half that in those on cyclosporine+steroid therapy, and no severe case was observed in the former, both groups of patients having received no anti-T cell globulin (see Bia M. J., et al., Transplantation, Vol. 40, pp. 610-614, "Effect of treatment with cyclosporine versus azathioprine on incidence and severity of cytomegalovirus infection post-transplantation" (1985)).
Development of CMV infection is largely influenced by the immune condition of the host, the degree of immune suppression, and the like and does not seem to be decided simply by the combination of immunosuppressants. Nevertheless, it is understood that the clinical observations of post-transplantation patients account for the influences of immunosuppressants on CMV proliferation in vitro more duly than expected.
The above situation implies possibility to alleviate CMV infectious disease by use of some of drugs currently employed as immunosuppressants which exhibit mild, while not potent, antiviral activity.
The reason why immunosuppressants essentially having weaker anti-CMV activity than general antiviral agents eventually exhibit effective anti-CMV activity will be explained below. Taking CMV-caused pneumonia for instance, it takes about 2 weeks for the very early stage (in which the X-ray picture of the chest demonstrates changes which retrospectively appear abnormal) to develop into the stage which is clinically recognized as CMV penumonia. Tentatively setting the doubling time of CMV in the body at 72 hours (3 days), CMV increases 4 to 5 times within 2 weeks. Assuming that mizoribine or azathioprine controls CMV proliferation to half as described above, the amount of the virus will be controlled by administering mizoribine or azathioprine to one-thirty second through the 5-fold doubling period, i.e., about 3% of the amount of the virus in the case of using no mizoribine or azathioprine. It is considered natural that no CMV-caused disease occurs with such a small amount of CMV in patients on mizoribine or azathioprine therapy.
On the other hand, FK 506 hardly affects CMV proliferation, while cyclosporine accelerates CMV proliferation about twice. Accordingly, the CMV amount in the case of cyclosporine therapy will be 32 times that in the case of FK 506 therapy as calculated in the same manner as above. In addition, FK 506 is concentrated in the lung, the target organ of CMV infection, and the CMV proliferation is suppressed at that concentration by FK 506. This fact appears to contribute to the difference between FK 506 therapy and cyclosporine therapy.
Thus, it has now been revealed that use of an immunosuppressant having weak but effective anti-CMV activity possibly alleviates CMV infectious disease. Conventional studies have never been directed to the relationship between CMV and immunosuppressants from this point of view.
On the other hand, traditional medicines (such as traditional Chinese medicines and traditional Japanese medicines) (it is called crude drugs hereinafter) have been used for therapy for a long number of years, and ample knowledge of their usage, dosage, etc. has been accumulated. Many of crude drugs have minor or substantially no side effects. However, studies on antiviral activity of crude drugs are rare. The literature on this subject now available includes Ito M., et al., Antiviral Research, Vol. 7, pp. 127-137, "Inhibitory effect of glycyrrhizin on the in vitro infectivity and cytopathic activity of the human immunodeficiency virus [HIV (HTLV-III/LAV)]", (1987); Takechi M. and Tanaka Y., Planta Medica, Vol. 42, pp. 69-74, "Purification and characterization of antiviral substance from the bud of Syzygium aromaticum", (1981); Kane C. J. M., et al., Bioscience Reports, Vol. 8, pp. 85-94, "Methyl gallate, methyl-3,4,5-trihydroxybenzoate, is a potent and highly specific inhibitor of herpes simplex virus in vitro: I. Purification and characterization of methyl gallate from Sapium sebiferum", (1988); Nagai T., et al., Chem. Pharm. Bull., Vol. 38, pp. 1329-1332, "Inhibition of influenza virus sialidase and anti-influenza virus activity by plant flavonoids", (1990); and Arai Y., et al., J. of Medical and Pharmaceutical Society for WAKAN-YAKU, Vol. 4, pp. 402-403, "Effect of Bezoar bovis against Chikungunya virus", (1987).